Preparation method of insect cell extract solution for cell-free protein synthesis, the insect cell extract solution and cell-free synthesis method of protein using the insect cell extract solution

a technology of insect cell extract and insect cell, which is applied in the field of preparation of insect cell extract solution for cell-free protein synthesis, the cell-free synthesis method of protein using the insect cell extract solution, can solve the problems of complex operation, large amount of protein synthesized using the extract solution obtained by this method, and the need for complicated manipulations. to achieve the effect of easy preparation

Active Publication Date: 2008-01-29
SHIMADZU CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007]The present invention has been made to solve the above-mentioned problems and aims at providing a preparation method of an insect cell extract solution for cell-free protein synthesis, which solution is easy to prepare and affords synthesis of a p...

Problems solved by technology

However, viable cells show a propensity toward elimination of exogenous proteins for their functional retention, and there ate many proteins that cannot be expressed easily since expression of cytotoxic proteins in viable cells prevents cell growth.
This method nevertheless requires special devices and tools for extraction from the insect cell, and the operation is complicated.
Moreover, complicated manipulations are necessary fo...

Method used

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  • Preparation method of insect cell extract solution for cell-free protein synthesis, the insect cell extract solution and cell-free synthesis method of protein using the insect cell extract solution
  • Preparation method of insect cell extract solution for cell-free protein synthesis, the insect cell extract solution and cell-free synthesis method of protein using the insect cell extract solution
  • Preparation method of insect cell extract solution for cell-free protein synthesis, the insect cell extract solution and cell-free synthesis method of protein using the insect cell extract solution

Examples

Experimental program
Comparison scheme
Effect test

reference example 1

Culture of Insect Cells

[0094]High Five (manufactured by Invitrogen) insect cells (2.1×107 cells) were cultured in a culture flask (600 cm2) containing an Express Five serum-free medium (manufactured by Invitrogen) supplemented with L-glutamine at 27° C. for 6 days. As a result, the cell count reached 1.0×108 and wet weight 1.21 g.

example 1

Preparation of Insect Cell Extract Solution

[0095]The insect cells cultured in the above-mentioned Reference Example 1 were collected, washed (centrifugation at 700×g, 4° C., 10 min) 3 times with a solution for extraction having the following composition and then suspended in 1 mL of a solution for extraction.

[0096]

[Composition of solution for extraction]  60 mMHEPES-KOH (pH 7.9) 200 mMpotassium acetate  4 mMmagnesium acetate  4 mMDTT 0.5 mMPMSF

[0097]This suspension was rapidly (within 10 sec) frozen in liquid nitrogen. After freezing sufficiently, the suspension was thawed in a water bath at about 10° C. After complete thawing, it was subjected to centrifugation at 15000×g, 4° C. for 15 min (himacCR20B3, manufactured by Hitachi Koki) and the supernatant was recovered. The recovered supernatant (1.5 mL) was applied to PD-10 desalting column (manufactured Amersham Biosciences) equilibrated with a buffer solution for gel filtration having the following composition.

[0098]

[Composition of...

reference example 2

Preparation of Exogenous mRNA

(1) Construction of Vector DNA

[0100]Using pGEM-luc vector (5 ng, manufactured by Promega) as a template, a primer (Luc T7-F3-Kpn) having a base sequence depicted in SEQ ID; No 1, a primer (Luc T7-R4-Kpn) having a base sequence depicted in SEQ ID; No 2, and KOD plus (manufactured by TOYOBO Co.), PCR was performed at 97° C., 15 sec, 55° C., 30 sec and 68° C., 120 sec, for 30 cycles. DNA fragment was purified by ethanol precipitation, and digested with KpnI.

[0101]Separately, pTNT Vector (manufactured by Promega) was digested with KpnI. These reaction solutions were separated by agarose gel electrophoresis, and using a Gen Elute Gel Purification Kit (manufactured by SIGMA), DNA fragment was purified. Using Ligation High (manufactured by TOYOBO Co.), these DNA fragments were ligated and Escherichia coli DH5α (manufactured by TOYOBO Co.) was transformed. Plasmid DNA was prepared from the transformed Escherichia coli by alkali-SDS methods, and subjected to a se...

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Abstract

The present invention provides a preparation method of an insect cell extract solution for cell-free protein synthesis, the insect cell extract solution, a protein synthesis method in a cell-free system, which uses the insect cell extract solution, and a kit for cell-free protein synthesis containing the insect cell extract solution. The extract solution is easily prepared by the method of the present invention and can synthesize a higher amount of protein than by extract solutions prepared by conventional methods.

Description

TECHNICAL FIELD OF THE INVENTION[0001]The present invention relates to a method for preparing an insect cell extract solution usable for cell-free protein synthesis, said insect cell extract solution, a cell-free synthesis method of a protein using said insect cell extract solution, and a kit for cell-free protein synthesis, containing said insect cell extract solution.BACKGROUND OF THE INVENTION[0002]In recent years, genetic information of many organisms, such as human genome, has been decoded. Under the circumstances, functional analysis of proteins and creation of genomic medicine based on such genetic information have been attracting attention for postgenomic studies. Application and utilization of proteins corresponding to such genetic information for pharmaceutical products and the like requires easy synthesis of extensive kinds of proteins in a short time.[0003]At present, expression systems using viable cells (hereinafter sometimes to be referred to as “cell-system”) of yeas...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12P21/06C12N1/06C12P21/00
CPCC12N1/066C12P21/00
Inventor EZURE, TORUHIGASHIDE, SHOKENSUZUKI, TAKASHIITO, MASAAKIENDO, KOKI
Owner SHIMADZU CORP
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